The present invention relates to a solid medium or support for use in the storage (preferably the long term storage) of nucleic acids (e.g. DNA and RNA, ribosomal RNA and messenger RNA), particularly one or more vectors and especially one or more plasmids, and to methods which comprise the use of this solid medium or support. In particular, the invention relates to a method for storage and transport of such nucleic acids or vectors on the solid medium, as well as to methods which involve either recovery or purification of the nucleic acids or vectors from the solid medium, or the use or manipulation of the nucleic acids or vectors obtained from or contained by or on the solid medium. Such use or manipulation includes, for example, digestion (e.g. with one or more nucleases, exonucleases or endonucleases such as restriction enzymes), synthesis (e.g. with one or more polymerases and/or reverse transcriptases), amplification (e.g. by polymerase chain reaction with one or more polymerases), sequencing (e.g. with one or more polymerases), or transformation or transfection into one or more host cells using, for example, chemically competent or electrocompetent cells or using known transfection reagents and techniques. The preferred medium or support is a matrix which protects against degradation of nucleic acid incorporated onto the matrix. Such a matrix may comprise an absorbent cellulose-based matrix or paper, or a micromesh of synthetic plastic material such as those described in U.S. Pat. No. 5,496,562, which is incorporated by reference herein. Preferably, the matrix comprises a composition comprising a weak base, a chelating agent, an anionic surfactant or anionic detergent, and optionally uric acid or a urate salt, wherein said composition is absorbed on or incorporated into said matrix. FTA® paper brand cellulose based solid matrix, and derivatives, variants and modifications thereof are included among such supports.
Another aspect of the invention relates to such a solid medium or support for use in the sampling, purifying and/or analyzing of one or more non-chromosomal DNA (e.g. mitochondria, chloroplast, F′) and to methods which comprise the use of this solid medium or support for such purposes.
For many projects, generation of numerous DNA samples from biological specimens is routine. Handling and archiving a large collection can become a logistical problem for the laboratory. One solution, used in forensic labs, is the blood-storage medium cellulose based solid matrix paper card, such as an FTA® Card. A cellulose based solid matrix paper card stores genomic DNA in the form of dried spots of human whole blood, the cells of which were lysed on the paper. Stored at room temperature, genomic DNA on a cellulose based solid matrix paper is reported to be stable, e.g., for at least 7.5 years using an FTA® Card brand cellulose based solid matrix (Burgoyne, et al., Conventional DNA Collection and Processing: Disposable Toothbrushes and FTA® Paper as a Non-threating Buccal-Cell Collection Kit Compatible with Automatable DNA Processing, 8th International Symposium on Human Identification, Sep. 17-20, 1997). Before analysis of the captured DNA, a few simple washing steps remove the stabilizing chemicals and cellular inhibitors of enzymatic reactions. Since the DNA remains with the paper, the manipulations to purify the DNA are simplified and amenable to automation. DNA samples on cellulose-based solid matrix cards offer a very compact archival system compared to glass vials or plastic tubes located in precious freezer space.
Bacterial DNAs spotted on cellulose based solid matrix cards may be an efficient system for storage and retrieval as well. Recently, Rogers and Burgoyne characterized by PCR-ribotyping culture samples of several bacterial strains of Staphylococcus and E. coli stored on FTA® Cards brand cellulose based solid matrix (Rogers, et al., (1997) Anal. Biochem. 247:223). Further, purified plasmid DNA was efficiently recovered after spotting on treated paper, however, encasement of the paper in polystyrene was used for storage (Burgoyne, U.S. Pat. No. 5,496,562, which is incorporated by reference herein).
According to the present invention, any nucleic acids (e.g. RNA and DNA and particularly RNA and DNA vectors) may be archived and later recovered and/or manipulated by a simple and efficient method in which the host carrying the one or more nucleic acids or vectors are contacted with a solid medium (preferably FTA® paper brand cellulose based solid matrix or derivatives, variants or modifications thereof), without the need to encase the support or use protective coatings (such as polystyrene) to store the nucleic acids or vectors. Thus, the invention avoids the need to use organic solvents or harsh chemicals to remove such protective coatings before use, manipulation, purification or isolation of the nucleic acid molecules or vectors can take place. In another aspect, purified nucleic acid molecules or vectors may be used, although in a preferred aspect, crude preparations (unpurified vector preparations) containing the one or more nucleic acid molecules or vectors may be contacted with the solid medium or support. Thus, the invention provides methods to isolate and/or purify nucleic acid molecules or vectors from any sample containing one or more vectors such as host cells, viruses, viral plaques, and/or crude preparations from biological materials (such as host cell or virus extracts, lysates, debri, hydrolysates, and the like). Such isolated and/or purified nucleic acid molecules or vectors obtained from or contained by the solid support or matrix may be used or manipulated in one or more standard molecular biology techniques, such as digestion, sequencing, amplification, synthesis and transformation/transfection reactions. In a particularly preferred aspect, one or more host cells containing the nucleic acid molecules or vectors to be isolated, stored and/or manipulated can be contacted directly with the medium or support. According to the present invention, host cell cultures or colonies from plates may be used. Preferred host cells for use in the invention include prokaryotic or eukaryotic host cells, particularly gram positive and gram negative bacteria such as Escherichia, Streptomyces, Pseudomonas, and the like.
Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of what is known in the art, in light of the following drawings and description of the invention, and in light of the claims.